PROJECT SUMMARY Natural products (NPs) continue to inspire novel chemistry, biology, and medicine, but the rate of discovery of novel NPs has slowed dramatically as most gene clusters encoding their biosynthesis are silent when the microorganisms are cultured under standard laboratory conditions. In this MIRA application, we propose to discover new NPs by mining actinomycetal genomes, develop enabling technologies to produce the target NPs in enough quantities for structural elucidation and biological evaluation, and characterize their biosynthetic machineries to discover new chemistry and enzymology. Our hypotheses have been: (i) genome survey of the actinomycetale strain collection at The Scripps Research Institute (TSRI) will allow us to identify potential producers of the targeted NP scaffolds, (ii) genome sequencing of the potential producers for the targeted biosynthetic gene clusters (BGCs) will allow us to predict the structural novelty of the new NPs, (iii) genetic manipulation of the most promising BGCs in their native producers or expression of them in heterologous model hosts will allow us to produce and isolate the new NPs, and (iv) characterization of the biosynthetic machinery of these new NPs will allow us to discover new chemistry and enzymology. Studies from two past and two current NIGMS grants have cumulated into (i) the construction of the Natural Products Library at TSRI that currently consists of ~211,000 microbial strains, ~461,000 crude extracts and partially purified fractions prepared from selected strains, and ~600 pure NPs, (ii) the establishment of C-1027 and tiancimycin as model systems for enediyne NP biosynthesis, leinamycin and guangnanmycin as model systems for biosynthesis of the leinamycin family of NPs (i.e., hybrid peptide-polyketide macrolactams featuring unprecedented sulfur functionalities), and platensimycin and platencin as model systems for bacterial diterpenoid and thiocarboxylic acid-containing/derived NP biosynthesis, respectively, and (iii) the identification of 190, 43, 66, and 264 distinct BGCs, encoding new enediynes, new members of the leinamycin family, new bacterial diterpenoids, and new thiocarboxylic acid-containing/derived NPs, respectively. These findings set the stage in the current application to mine the actinomycetal genomes for NP discovery and biosynthesis. The outcomes of this application include (i) fundamental contributions to genome mining and activation of large BGCs, in native producers and heterologous model hosts, for NP production and structural diversity by metabolic pathway engineering, (ii) discovery of new NPs, with privileged scaffolds, to inspire new chemistry, biology, and medicine, and (iii) new insights into biosynthetic machineries and novel chemistry and enzymology for the biosynthesis of enediynes, the leinamycin family of NPs, bacterial diterpenoids, and thiocarboxylic acid- containing/derived NPs. The long-term goal of our research is to understand at a molecular level how microorganisms synthesize complex NPs and to exploit this knowledge to discover novel NPs and engineer their analogues for drug discovery.